KR20180135912A - Compositions and methods for stabilizing alphaviruses using improved formulations - Google Patents

Abstract

Embodiments herein are directed to compositions and methods for stabilizing live alphaviruses. Other embodiments are directed to compositions and methods for reducing the degradation of live, attenuated alpha viruses. Certain embodiments relate to providing a stabilizing composition while reducing the immune response in a subject to an excipient that stabilizes the live alpha virus by providing an improved formulation. Yet another embodiment relates to a composition for reducing the degradation of said live alphavirus and the use of the compositions disclosed herein in a kit for portable applications.

Description

Compositions and methods for stabilizing alphaviruses using improved formulations

preference

This PCT application claims the benefit under 35 USC § 119 (e) of U.S. Provisional Patent Application Serial No. 62 / 316,262, filed on March 31, 2016, the disclosure of which is incorporated herein by reference.

Government support

This invention was made with government support under authorization R01AI093491 granted by the National Institutes of Health. The government has certain rights in the invention.

Field

Embodiments herein are directed to compositions and methods for stabilizing alphaviruses. In certain embodiments, the compositions and methods disclosed herein relate to the stabilization of live alpha viruses or the stabilization of live, attenuated alpha viruses. Other embodiments are directed to compositions and methods for reducing the degradation of alpha viruses. Still other embodiments are methods of treating a living, attenuated or live alphavirus with a reduced immune response in a subject to an excipient by providing an improved formulation for stabilization and delivery of the compositions disclosed herein to a subject, To providing formulations to live alpha viruses. Other embodiments relate to formulations for mannitol, histidine and sucrose in human or other mammal-derived by-products such as HEPES buffer without protein. Yet another embodiment relates to an alpha viral formulation for use in a kit for portable applications and methods.

Vaccines for protection against viral infections have been effectively used to reduce the incidence of human or animal viral diseases. One of the most successful techniques for antivirus is to immunize an animal or human with a weakened or attenuated strain of the target virus ("live, attenuated virus"). Due to limited replication after immunization of the subject, attenuated strains do not cause viral-related disease because they can not overlap and pass on the stomach. However, attenuated viruses can still express viral antigens and produce a potent and long lasting immune response to the virus in exposed subjects. Thus, upon subsequent exposure to the virus, the immunized individual has a reduced chance of developing the disease associated with the viral infection.

To ensure that live, attenuated viruses are effective in vaccines, live, attenuated viruses must be able to replicate after immunization and the virus itself must be protected from degradation to be used effectively and accurately. Some vaccines are sensitive to temperature extremes; Either excessive heat or accidental freezing can deactivate the virus. Maintaining efficacy and protective environment throughout distribution is particularly difficult when treating patients in developing countries or in remote areas.

Embodiments herein are directed to compositions and methods for stabilizing live, alpha viruses. Other embodiments are directed to compositions and methods for stabilizing and / or reducing degradation of live, attenuated alpha viruses used in immunogenic compositions for immunizing a subject against infection caused by an alpha virus. Another embodiment is directed to providing a stabilizing composition that does not contain human or other mammalian by-products to reduce the immune response in a subject to an excipient that stabilizes live, attenuated alpha viruses. According to these embodiments, the stabilized formulation does not contain animal by-products when used as an immunogenic composition against alpha virus infection in the subject. Still other embodiments relate to the use of the stabilizing compositions disclosed herein in kits for portable applications and methods.

In some embodiments, certain compositions disclosed herein may comprise a combination of ingredients that, when administered to a subject, reduce the degradation of the live, attenuated alpha virus while providing a formulation with a reduced allergic response by the subject have. The specific formulations disclosed herein are designed to provide a higher level of safety of the immunogenic alpha virus formulations by taking into account the purity of the raw materials used to stabilize the alpha viruses used in the immunogenic alpha viral formulations. Some formulations in the past use animal-derived excipients and other agents for virus stabilization. In certain circumstances, some of these animal-derived excipients and proteins may carry heterogeneous agents (e. G., BSE or allergic antigens) that can cause adverse reactions in the subject when exposed to these agents. Formulations without human or other mammal-derived excipients reduce the risk of introducing unknown or unstable agents (e. G., Impurities associated with these agents) into the subject. In addition to stabilizing animal-derived formulation-containing formulations, or better animal-derived formulation-free formulations are provided herein. Other embodiments disclosed herein relate to combinations of excipients that greatly enhance the stability of live, attenuated alpha viruses. Additional embodiments include methods for reducing the need for lower temperatures ( e. G. Refrigerated or frozen storage) to facilitate stabilized transfer of virus formulations in the ozone layer and for stabilizing the stability of the virus formulation ≪ RTI ID = 0.0 > and / or < / RTI > In other embodiments, the compositions and methods disclosed herein can be used to reduce the resumption time of the composition and / or improve physiological properties of the composition as compared to other formulations that do not contain these compositions, as well as the storage of aqueous and / or reconstituted live alphavirus Life span.

In certain embodiments, the formulations disclosed herein can confer a stabilizing effect from the loss of viral titers in liquid, frozen, lyophilized and re-hydrated formulations. In other embodiments, the compositions disclosed herein are administered to a living or attenuated alphavirus composition comprising, without limitation, one or more live alphaviruses, HEPES buffer, one or more amino acids, and one or more carbohydrate agents . In other embodiments, the stabilizing compositions disclosed herein may comprise one or more carbohydrate preparations comprising a living alphavirus, an amino acid comprising histidine, and one or more sucrose and mannitol in HEPES buffer. In yet another embodiment, a live alpha viral formulation can comprise an alpha virus, living in a HEPES-based buffer with histidine, mannitol, and sucrose.

In some embodiments, the alpha viral formulations can include, but are not limited to, one or more live, attenuated alpha viruses. According to these embodiments, one or more live, attenuated alpha viruses may be combined in buffers with mannitol, histidine, and / or sucrose. In certain embodiments, the buffer, e. G., The base buffer, may comprise a HEPES buffer. According to these embodiments, the buffered HEPES in a live, attenuated alpha virus composition may be about pH 6.0 to about pH 10.0 with a concentration of about 1.0 to about 200.0 mM HEPES, and the histidine may be in a concentration from about 0.1% to about 5.0 (w / v) (about 1 to about 50 mg / ml), sucrose can be about 0.1% to about 20% (w / v) Can be from about 1.0% to about 15% (w / v) (about 1 to about 150 mg / mL). In other embodiments, the HEPES buffer concentration may be from about 5.0 mM to about 50.0 mM, from about 10.0 mM to about 30 mM, or from about 10.0 mM to about 20 mM.

In certain embodiments, in a live attenuated alpha virus composition, the alphavirus formulation buffer can be about 15 mM HEPES buffer at a pH of about 6.5 to about 8.0, about 7.0 to about 7.5, or about 7.2, wherein the histidine is about 5.0 mg / ml to about 20.0 mg / ml, sucrose can be about 35.0 mg / ml to about 50.0 mg / ml, and mannitol can be about 25.0 mg / ml to about 40.0 mg / ml. In other embodiments, the formulations disclosed herein for stabilizing live alpha viruses or live attenuated alpha viruses may comprise a HEPES buffer having a concentration of about 15.0 mM solution at pH 7.2 while the histidine is about 5.0 mg / mL to about 12.5 mg / mL, sucrose can be about 35.0 mg / mL to about 50.0 mg / mL, and mannitol can be about 25.0 mg / mL to about 40.0 mg / mL.

In other embodiments, one or more live attenuated alphaviruses may be used to stabilize live attenuated alpha viruses under various conditions, including but not limited to sucrose, sodium chloride, monosodium glutamate, sodium phosphate, potassium phosphate, and potassium chloride ≪ RTI ID = 0.0 > and / or < / RTI > According to these embodiments, the HEPES buffer may be, but is not limited to, sucrose having a concentration of from about 1.0% to about 10.0% (w / v); Sodium chloride having a concentration of about 0.05% to about 1.5% (w / v); Monosodium glutamate having a concentration from about 0.05% to about 0.5% (w / v); Sodium phosphate having a concentration of about 0.01 to about 0.15% (w / v); Potassium phosphate having a concentration from about 0.01% to about 0.05% (w / v); And potassium chloride having a concentration of from about 0.005% to about 0.05% (w / v) in combination with a live alpha virus or a live, attenuated alpha virus in which the alpha virus is stabilized and the degradation of the alpha virus is reduced.

In another embodiment, the concentration of sucrose in combination with live alpha viruses in which live alpha viruses are stabilized, degradation is reduced, and shelf life is prolonged may be about 5.0% (w / v); Sodium chloride may be about 0.64% (w / v); Monosodium glutamate may be about 0.1% (w / v); Sodium phosphate may be about 0.09% (w / v); Potassium phosphate may be about 0.016% (w / v); Potassium chloride may be about 0.016% (w / v). Some embodiments disclosed herein are directed to methods for reducing inactivation of live alphaviruses. According to these embodiments, the method includes, but is not limited to, compositions comprising 0.1 mM to 200.0 mM HEPES buffer, one or more carbohydrate preparations (such as monosaccharides, sugar alcohols and other homologs), and one or more amino acids, , ≪ / RTI > According to these embodiments, when combined with a live, attenuated alpha virus, the HEPES buffer can be about pH 6.0 to about pH 9.0 at a concentration of about 1.0 mM to about 200 mM, and histidine can be about 0.1% to about 5.0% w / v), sucrose may be from about 0.1% to about 20% (w / v), and mannitol may be from about 1.0% to about 10% (w / v).

Some embodiments disclosed herein may relate to methods for reducing inactivation of live alphaviruses. According to these embodiments, the one or more live alpha viruses can be combined with a composition comprising, but not limited to, 0.1 to 200 mM HEPES buffer, one or more carbohydrate formulations, and one or more salt formulations. According to these embodiments, the sucrose concentration when combined with live alphavirus may be from about 1.0% to about 10.0% (w / v) to reduce degradation of the alpha virus; The sodium chloride concentration may be from about 0.1% to about 1.5% (w / v); The monosodium glutamate concentration may be from about 0.05% to about 0.5% (w / v); The sodium phosphate concentration may be from about 0.01 to about 0.15% (w / v); The potassium phosphate concentration may be from about 0.01% to about 0.05% (w / v); The potassium chloride concentration may be from about 0.01% to about 0.05% (w / v). In another embodiment, the sucrose concentration when combined with live or live, attenuated alpha virus can be about 5.0% (w / v); The sodium chloride concentration may be about 0.64% (w / v); The monosodium glutamate concentration may be about 0.1% (w / v); The sodium phosphate concentration may be about 0.09% (w / v); The potassium phosphate concentration may be about 0.016% (w / v); The potassium chloride concentration may be about 0.016% (w / v).

In certain embodiments, the compositions disclosed herein can be used to reconstitute or rehydrate lyophilized live alpha viruses. For example, after transport of a live, dehydrated or lyophilized or frozen live alpha virus from frozen to room temperature and other homogenous to various temperatures. The compositions used herein can reduce the inactivation and / or degradation of live and live, attenuated alpha viruses at various storage temperatures and during a freeze-thaw cycle and / or increase stabilization.

In certain embodiments, live or live attenuated alpha viruses that may benefit from the compositions disclosed herein include, but are not limited to, chironoquine virus (CHIK), sanguine Viruses, Ross River virus, eastern encephalitis, Venezuelan equine encephalitis virus, and western encephalitis or other alphaviruses. Other Semliki forest heat virus complexes include, but are not limited to, Bevarovirus, Mayovirus, Subtypes: Unavirus, Sourgan Viruses: Subtype: ; Subtype: Geta virus; Subtypes: Sagiyama virus, and Semliki forest heat virus: Subtype: Metry virus.

Other embodiments disclosed herein are directed to live, attenuated alphavirus compositions and methods for vaccine or immunogenic composition creation that are capable of reducing or preventing the onset of a medical condition caused by one or more alpha viruses as contemplated herein . In certain embodiments, one or more live, attenuated viruses of the compositions contemplated herein may be part of a pharmaceutical composition and include pharmaceutically acceptable excipients. The pharmaceutical compositions disclosed herein can be used to reduce the onset of an infection with an alpha virus and conditions caused by an alpha virus infection when the pharmaceutical composition is administered to a subject. The subject may be a mammal, for example, a breed animal, a pet, a livestock, another animal or a human subject (adult or child).

Other implementations include, but are not limited to, containers; And kits for reducing the inactivation of live attenuated viruses comprising the compositions disclosed herein.

The following drawings form part of this specification and are included to further demonstrate certain aspects of certain implementations herein. Embodiments will be better understood with reference to one or more of these drawings in combination with the detailed description set forth herein.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an exemplary histogram plot showing experiments using various liquid and lyophilized compositions for stability testing of exemplary live alpha viruses in one embodiment disclosed herein.
Figure 2 is an exemplary histogram plot showing experiments using various liquid compositions for the stability testing of exemplary live alpha viruses in one embodiment disclosed herein after a freeze-thaw process.
Figure 3 is an exemplary graph illustrating experiments using various compositions for testing the stability of exemplary live alpha viruses in one embodiment disclosed herein at about 40 ° C.
Figure 4 is an exemplary graph illustrating experiments using various compositions for testing the stability of exemplary live alpha viruses in one embodiment disclosed herein at about 25 ° C.
Figure 5 is an exemplary graph depicting experiments using various compositions for testing the stability of exemplary live alpha viruses in one embodiment disclosed herein at about 4 < 0 > C.
Figure 6 is a histogram plot showing a cumulative summary of experiments using various compositions for testing the stability of exemplary live alpha viruses in one embodiment disclosed herein at various temperatures.
Figure 7 is a histogram showing the concentration range of the various agents used in one embodiment disclosed herein in which the formulations included in these exemplary compositions for stabilizing live alpha viruses are mannitol, histidine, and sucrose.
Figure 8 is a histogram showing experiments using various compositions disclosed herein in liquid and lyophilized conditions for effect testing in live alpha viruses in one embodiment disclosed herein.
Figure 9 is a histogram showing experiments using various compositions disclosed herein under freeze-thaw conditions for effect testing in live alpha viruses in one embodiment disclosed herein.
Figure 10 is an exemplary graph depicting an experiment for stability testing of live alpha viruses in various formulations in one embodiment disclosed herein at about 40 ° C.
Figure 11 is an exemplary graph depicting an experiment for stability testing of live alphaviruses in various formulations in one embodiment disclosed herein at about 25 캜.
Figure 12 is an exemplary graph depicting experiments for stability testing of exemplary live alpha viruses in various formulations in one embodiment disclosed herein at about 4 캜 or about refrigeration temperature.
Figure 13 is a histogram plot showing a cumulative summary of experiments using various compositions for the stability testing of example live, attenuated alpha viruses in one embodiment disclosed herein at various temperatures.
Figure 14 is a graph of various formulations under different conditions compared to the formulation itself and other formulations used for predicting the preferred stabilizing composition for live, attenuated alpha viruses in one embodiment disclosed herein.
Figure 15 is a histogram showing the concentration range of the various agents used in one embodiment disclosed herein in which the formulations included in these exemplary compositions for live alpha virus stabilization are mannitol, histidine, and sucrose.
Figure 16 is a histogram showing experiments using various compositions disclosed herein in liquid and lyophilized conditions for effect testing in live alpha viruses in one embodiment disclosed herein.
Figure 17 is an exemplary graph showing an experiment for stability testing of live alpha viruses in various formulations in one embodiment disclosed herein at about 40 < 0 > C.
Figure 18 is an exemplary graph depicting experiments for stability testing of live alpha viruses in various formulations in one embodiment disclosed herein at about 25 캜.
19 is an exemplary graph depicting an experiment for stability testing of example live alpha viruses in various formulations in one embodiment disclosed herein at about 4 DEG C or about refrigeration temperature.
Figure 20 is a histogram plot showing a cumulative summary of experiments using various compositions for the stability testing of exemplary live, attenuated alpha viruses in one embodiment disclosed herein at various temperatures.

Justice

As used herein, " one " or " one " may mean one or more than one of the items.

As used herein, " about " can mean maximum and plus or minus 5 percent, for example, about 100 can mean 95 and up to 105.

As used herein, CHIKV can refer to Chikunguniya virus.

As used herein, TCID50 can mean a 50% tissue culture infectious dose.

As used herein, HB can refer to HEPES buffer saline.

As used herein, HBS can mean HEPES buffer saline + sucrose.

As used herein, DMEM can mean Dulbecco's modified minimal essential medium.

As used herein, PBS may refer to phosphate buffered saline.

As used herein, FBS may refer to fetal bovine serum.

As used herein, Lyo may refer to lyophilized or dehydrated depending on the formulation of the reference.

As used herein, " attenuated alpha virus " or " live, attenuated alpha virus " refers to a vaccine that demonstrates reduced or no clinical signs of disease when administered to a subject It can mean alpha virus.

details

In the following sections, various illustrative compositions and methods are described in order to describe various embodiments in detail. It is to be understood that various implementations of the practice do not require the use of all or even some of the specific details described herein, rather that concentrations, times, and other specific details may be modified through routine experimentation It will be obvious to the skilled person. In some instances, well-known methods or ingredients have not been included in the description.

In certain embodiments, the stability of live, attenuated alpha viruses used in immunogenicity or vaccine formulations has been observed in the various formulations disclosed herein. In some embodiments, formulations for increasing alpha virus stability by reducing loss of potency in liquid, frozen, lyophilized and re-configured or re-hydrated live, attenuated alpha viruses have been demonstrated. In certain embodiments, live alpha viral formulations can include, but are not limited to, one or more live or live, attenuated alpha viruses, HEPES buffer, one or more amino acids, and one or more carbohydrate agents. In certain embodiments, the compositions disclosed herein may comprise one or more carbohydrate preparations comprising an alphavirus, at least one amino acid comprising histidine, and at least one sucrose and / or mannitol in a HEPES buffer. Certain compositions disclosed herein can include a salt or salt solution. According to these embodiments, the formulations disclosed herein may be used in liquid, frozen or lyophilized storage of attenuated alpha viruses that live or live at or above about-80 C to about 40 C without significant loss of alphavirus titer in the composition Can be used. In some embodiments, for example, long term storage at 4 DEG C can be achieved using the formulations disclosed herein. In certain embodiments, the compositions contemplated herein may be dehydrated or hydrated, either partially or wholly.

In some embodiments, live alpha viruses or live attenuated alpha viruses that are considered beneficial from the compositions disclosed herein may be used in coronaviridae and in the serogroups, including, but not limited to, chiculonya virus (CHIK) Sour virus, Ross River virus, eastern encephalitis, Venezuelan equine encephalitis virus and western encephalitis or other alphaviruses. Other Semliki forest heat virus complexes include, but are not limited to, Bevarovirus, Mayovirus, Subtypes: Unavirus, Sourgan Viruses: Subtype: ; Subtype: Geta virus; Subtypes: Sagiyama virus, and Semliki forest heat virus: Subtype: Metry virus.

Certain embodiments disclosed herein relate to Chicken Growth Virus. Chikunguniya virus is an alpha virus with a positive sense single-stranded RNA genome of approximately 11.6 kb. It is a member of the Semliki forest heat virus complex and is closely related to Ross River virus, Sourgan virus and Semliki forest heat virus. The compositions disclosed herein can be used to stabilize the virus of the Semliki Forest Thermovirus complex variant by reducing the degradation of living alpha viruses or live, attenuated alpha viruses.

In certain embodiments, the live attenuated alpha viruses contemplated herein may comprise a chimeric alpha virus. In other embodiments, the live attenuated alphavirus may comprise live attenuated or chimeric chiquenginavirus. According to these embodiments, one exemplary chimeric alpha virus is one that can be expressed in mammalian cells and not in insect cells. In certain embodiments, live, attenuated alpha viruses can include live attenuated alpha viruses that can replicate in mosquito and mosquito cells. In other embodiments, the live attenuated alpha viruses considered to be beneficial from the compositions disclosed herein may be engineered to be under a eukaryotic control (e. G., The insertion of an eukaryotic component such as an IRES sequence and / Inactivation of the subgenomic promoter). In another embodiment, the live attenuated alpha virus comprises (i) an insertion of an internal ribosome entry site (IRES) between the end of the unstructured protein 4 (nsP4) coding sequence and the initiation AUG of the subgenomic RNA coding sequence of the alphavirus , And (ii) an inactivated subgenomic promoter, wherein the alpha virus is attenuated. In certain embodiments, the IRES sequence may be a brain myocarditis virus (EMCV) IRES or other similar IRES capable of creating a viable attenuated alpha viral construct used in a vaccine composition.

Embodiments herein are directed to methods and compositions for reducing or preventing the degradation and / or inactivation of live, attenuated alpha viruses. According to these embodiments, certain compositions may comprise a combination of components that reduce the degradation and / or inactivation of live, attenuated alpha viruses. Other embodiments herein relate to combinations of agents that can enhance the stability of live, attenuated alpha viruses. Still other compositions and methods herein include the need for lower temperatures ( e.g., refrigerated or frozen storage) while increasing the shelf life of aqueous and / or reconstituted living alpha viruses or live, attenuated alpha viruses .

In some embodiments, one or more live alpha viruses or live, attenuated alpha viruses can be combined with one or more amino acids and one or more carbohydrates. In certain embodiments, the alpha viral formulations disclosed herein may comprise at least three components of HEPES buffer, carbohydrate (s) and amino acid. In other embodiments, formulations for stabilization of live alpha viruses disclosed herein may include compositions that do not contain human or mammalian formulations, such as proteins ( e. G. , Serum-free or gelatin). In other embodiments, salt formulations may be added to these compositions to enhance the buffering capacity or other desired properties of the formulations.

Other embodiments disclosed herein are directed to a method of treatment of a living alpha virus or a live virus or a live virus for use in the preparation of an immunogenic or vaccine composition capable of reducing or preventing the side effects of exposure caused by one or more alpha viruses , And to attenuated alpha virus compositions. Subjects that are considered beneficial from these immunogenic or vaccine compositions include humans (e.g., adults, adolescents, children and infants) and animals such as pets, livestock or other raised or wild animals.

Some embodiments include, but are not limited to, one or more protein preparations, one or more amino acids; A composition capable of reducing the inactivation of an alpha virus comprising one or more carbohydrates, one or more saccharides or polyol formulations, and optionally, a HEPES buffer, and one or more alpha viruses or live, attenuated alpha viruses, , Or a method of reducing the inactivation of live, attenuated alpha viruses, wherein said composition reduces the inactivation of alpha viruses. In accordance with these embodiments, live attenuated viruses are characterized by having a feature similar to a particular alpha virus, such as chiquiniyya (e.g., Semliki Forestry Thermal Complex virus), and having a genetic structure similar to the alphavirus chiquiniya, And other alphaviruses having a non-structural protein layout and / or secondary structure or other homologues.

In some embodiments, the carbohydrate used in the formulations disclosed herein may be a sugar or a polyol or a sugar alcohol. According to these embodiments, the saccharide may be selected from, but not limited to, monosaccharides, such as glucose, galactose, ribose, mannose, rhamnose, talos, xylose, or alos arabinose, disaccharides such as trehalose, ( For example , acarbose, raffinose, mellyose, lecithin, or the like) or a salt thereof, or a salt thereof , if monohydrate, Panos, or cell Lot Rios) or polymeric sugar (for example dextran, xanthan, pullulan, cyclodextrin, amylose, amylopectin, starch, cells come logo sugars, cellulose, malto-oligosaccharide, glycogen, chitosan, Or chitin). The polyol may be selected from the group consisting of but not limited to sugar alcohols such as mannitol, sorbitol, arabitol, erythritol, maltitol, xylitol, glycitol, glycol, polyglycitol, polyethylene glycol, polypropylene glycol, isomalt, hydrogenated starch hydrosilate And glycerol.

In certain embodiments, other carbohydrate preparations for use in the formulations disclosed herein that may be considered for use in compositions herein include, but are not limited to, sucrose, fructose, galactose, trehalose, mannitol, sorbitol, amylose, Amylopectin, starch, cellulose, maltooligosaccharide, glycogen, chitosan, or chitin. In other embodiments, the amino acids contemplated for use herein include at least histidine and, optionally, glycine.

In some embodiments, the HEPES buffer in a live attenuated alpha virus composition can have a pH of about 6.0 to about pH 10, a concentration of about 1.0 mM to about 200 mM, and a concentration of at least one amino acid of about 1.0 mg / ml To about 40 mg / ml, and the at least one carbohydrate preparation concentration can be about 1.0 mg / ml to about 100 mg / ml. In other embodiments, the concentration of the HEPES buffer in the live, attenuated alpha virus composition may be about pH 6.5 to about pH 8.0 at a concentration of about 1.0 mM to about 40 mM, and the concentration of one or more amino acids may be about 1.0 mg / 30 mg / ml, and the at least one carbohydrate preparation concentration can be from about 10 mg / ml to about 50 mg / ml.

In certain embodiments, the live attenuated alpha virus composition may include, but is not limited to, HEPES buffer, mannitol, histidine, and sucrose. According to these embodiments, the HEPES buffer may be at a pH of about pH 6.0 to about pH 10.0 at a concentration of about 1.0 mM to about 200 mM, the histidine may be at a concentration of about 0.1% to about 5.0% The concentration can be from about 0.1% to 20.0%, and the mannitol can be from about 1.0% to about 10% concentration and one or more live, attenuated alpha viruses. In certain embodiments, the formulation may further comprise a 10 mM to 150 mM salt ( e.g., sodium chloride or other suitable salt).

Some embodiments of compositions and methods for reducing the inactivation of live, attenuated alpha virus compositions include, but are not limited to, compositions containing from 0.1 mM to 200 mM HEPES buffer, one or more carbohydrate preparations, and one or more amino acids Lt; / RTI > with one or live attenuated alpha virus. According to these embodiments, the concentration of buffered HEPES when combined with live, attenuated alpha virus may be about pH 6.0 to pH 10 at about 1 to about 200 mM, and histidine may be about 0.1% to about 5.0% , Sucrose can be from about 0.1% to about 20%, and mannitol can be from about 1.0% to about 10%. In some embodiments, the HEPES buffer when combined with a live, attenuated alpha virus can be about pH 6.0 to about pH 10 at about 1 to about 40 mM while the histidine is between about 5 mg / ml and about 50 mg / ml And sucrose can be about 5 mg / ml to about 50 mg / ml, and mannitol can be about 5 mg / ml to about 50 mg / ml. In other embodiments, the HEPES buffer when combined with a live, attenuated alpha virus can be from about pH 6.5 to about pH 7.5 at about 1 to about 30 mM, while the histidine can be from about 5 to about 30 mg / ml , Sucrose can be about 10 to about 50 mg / ml, and mannitol can be about 5 to about 40 mg / ml. In other embodiments, the HEPES buffer when combined with a live, attenuated alpha virus can be at about pH 7.2 at about 10 to 20 mM while the histidine can range from about 5 mg / ml to about 25 mg / ml, The sucrose can be about 20 mg / ml to about 50 mg / ml, and the mannitol can be about 10 mg / ml to about 40 mg / ml. In another embodiment, when combined with live attenuated alpha virus, the HEPES buffer may be a 15 mM solution at pH 7.2 while the histidine may be from about 5 mg / ml to about 20 mg / ml, From about 35 mg / ml to about 50 mg / ml, and mannitol from about 25 mg / ml to about 40 mg / ml. In another embodiment, the HEPES buffer when combined with live attenuated alphavirus may be a 15 mM solution at pH 7.2, wherein the histidine is between about 5.0 mg / ml and 12.5 mg / ml, the sucrose is about 35.0 mg / ml to about 50.0 mg / mL, and the mannitol is about 25.0 mg / mL to about 40.0 mg / mL.

Some embodiments disclosed herein include, but are not limited to, a non-limiting combination of a composition comprising at least one carbohydrate formulation, and at least one salt preparation, with from 0.1 mM to 200 mM HEPES buffer, at least one carbohydrate preparation, And methods for reducing the inactivation of live or attenuated alpha viruses, which may include live alpha viruses. According to these embodiments, the sucrose concentration may be from about 1.0% to about 10.0% (w / v) to reduce degradation of the alphavirus; The sodium chloride concentration may be from about 0.1% to about 1.5% (w / v); The monosodium glutamate concentration may be from about 0.05% to about 0.5% (w / v); The sodium phosphate concentration may be from about 0.01 to about 0.15% (w / v); The potassium phosphate concentration may be from about 0.01% to about 0.05% (w / v); The potassium chloride concentration may be from about 0.01% to about 0.05% (w / v). In another embodiment, the sucrose concentration when combined with live alpha virus can be about 5.0% (w / v); The sodium chloride concentration may be about 0.64% (w / v); The monosodium glutamate concentration may be about 0.1% (w / v); The sodium phosphate concentration may be about 0.09% (w / v); The potassium phosphate concentration may be about 0.016% (w / v); The potassium chloride concentration may be about 0.016% (w / v).

In certain embodiments, the compositions disclosed herein can be used to rehydrate partially or completely lyophilized live alphavirus or to deliver live alpha virus at various temperatures from various temperatures. The compositions contemplated herein may be used to reduce the inactivation and / or degradation of attenuated alpha virus and / or to increase stabilization during processing, manufacture or purification, during transport, during delivery, and during a freeze- thaw cycle at various storage temperatures .

In certain embodiments, the compositions contemplated herein may be dehydrated or hydrated, either partially or wholly. In addition, the compositions disclosed herein can be used during and after lyophilization of live alpha viruses. According to these embodiments, the composition comprises 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; over 90; Or 95% or more. The compositions described herein can increase the shelf life of aqueous or rehydrated live attenuated alpha viruses. In addition, the compositions disclosed herein can also be used in the treatment of a living, attenuated (non-toxic) condition at elevated temperatures, such as room temperature, subzero temperature, elevated temperature (e.g., from -80 캜 to 40 캜 and greater) under lyophilized or liquid / Thereby increasing the stability of the alpha virus. In other embodiments, the compositions disclosed herein can be used in compositions comprising HEPES buffer, one or more carbohydrate preparations, and one or more amino acid compositions that are 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times It is possible to increase the stability of live alpha viruses by more than 10 times.

In other embodiments, the methods disclosed herein may include lyophilization or other dehydration methods to preserve the alpha virus in the disclosed compositions. According to these embodiments, the compositions disclosed herein can reduce the inactivation of lyophilized or partially or totally dehydrated live alphaviruses. In another method, a composition for reducing inactivation of living alpha viruses may comprise dehydrated and rehydrated compositions or may comprise an aqueous composition. In certain embodiments, the compositions disclosed herein can increase the shelf life of aqueous or rehydrated alphavirus formulations by reducing degradation of the alphavirus.

In certain embodiments, compositions contemplated herein may be stored at room temperature ( e.g., from about 20 ° C to about 25 ° C, or even as high as 40 ° C) or at refrigeration temperatures ( such as from about 0 ° C to about 10 ° C, Can reduce the inactivation and / or degradation of live attenuated alpha viruses hydrated for more than 12 to over 24 hours. In some embodiments, the compositions disclosed herein can maintain an attenuated alpha virus that is 90% or more live for more than 24 hours. In addition, the compositions disclosed herein can reduce the inactivation of live alphaviruses hydrated during at least 2, at least 3, at least 4, at least 5, at least 6, and excess freeze-thaw cycles. Other embodiments are directed to compositions that are capable of reducing the inactivation of hydrated or aqueous live alpha viruses for greater than about 24 hours to 26 weeks at refrigeration temperatures (e.g., from about 0 ° C to about 10 ° C). Compositions contemplated in these methods may include, but are not limited to, HEPES buffer, histidine, sucrose, and mannitol. In certain embodiments, the live, alpha virus composition comprises greater than 90%, or greater than about 90%, or about 80% or about 70% viral titers and greater than 90% after about 4 weeks at a refrigeration temperature of about 4 & Maintain virus titers. Another embodiment herein is a pharmaceutical composition comprising about 90%, or about 80% virus or about 70% virus titer after twelve weeks at about 25 ° C and about 90%, or about 80% virus titer or about RTI ID = 0.0 > 70% < / RTI > Other embodiments contemplated herein are those in which the concentration of viral titer after about several hours ( e.g., 20 hours) at about 40 째 C as compared to other compositions known in the art is reduced to about 3 to about 10, Virus composition.

In other embodiments, the compositions contemplated herein may contain minor or univalent cations. For example, the compositions contemplated herein may have minor or no calcium / magnesium (Ca ⁺² / Mg ⁺² ).

Other live, attenuated viral formulations have been described previously (see for example Burke, Hsu et al. 1999). One common stabilizer, referred to as SPGA, is a mixture of 2-10% sucrose, phosphate, potassium glutamate and 0.5-2% serum albumin (see, for example, Bovarnick, Miller et al. 1950). Various modifications of these basic formulations have been identified with different cations, by the substitution of starch hydrolyzate or dextran for sucrose, and by the substitution of casein hydrolyzate or poly-vinylpyrrolidone for serum albumin. Other formulations use serum albumin as a protein source instead of hydrolyzed gelatin (Burke, Hsu et al. 1999). However, gelatin can cause allergic reactions in immunized children and adults and can be a cause of vaccine-related adverse events. In addition, gelatin may be supplied from the bovine or porcine bone and vertebral material. These sources have been sources of heterogeneous agents (retroviruses and / or prion-BSE) and provide safety concerns. In addition, albumin can be collected from humans - which also pose a potential risk of introducing an unhealthy heterogeneous agent. Thus, one goal disclosed herein is to provide a formulation having equivalent to an improved stabilizing effect in a live, attenuated or live alphavirus with no negative allergic response in the subject or possible safety concerns to the formulation, e.g., gelatin and / or serum albumin It was to confirm. In certain embodiments, the formulations disclosed herein can be compared to formulations containing gelatin that demonstrate that this formulation is equivalent to a superior stabilizing effect in the alpha virus without adverse effects of the gelatin-containing formulation.

Embodiments herein disclose compositions that reduce the chance of or reduce the aggravation of, or improve the stability of, a live alpha virus composition compared to that in the prior art. Certain compositions disclosed herein have a maximum of 1 day at about 40 < 0 >C; Up to 2 days; Provides stability of the aqueous virus for up to 4 days and over 6 weeks. Certain compositions disclosed herein provide stability of the aqueous virus for up to about 1 day to about 12 weeks at about room temperature ( e.g., 25 占 폚). Embodiments disclosed herein increase the protection of live viruses from, for example, freezing and / or thawing, and / or elevated temperatures. In certain embodiments, the compositions disclosed herein can stabilize live, attenuated viral products that have been dehydrated at room temperature conditions ( e.g., about 25 DEG C), reduce aging of the products, and / or prevent inactivation of the products. In other embodiments, the compositions contemplated herein can stabilize viral products that are aqueous live, at or above about 25 ° C or at or above about 40 ° C, to prevent degradation of the product and / or to inactivation of the product. The compositions disclosed herein can facilitate the storage, distribution, delivery and precise administration of alpha virus vaccines in developed and underdeveloped regions.

It will be understood by those skilled in the art that the compositions or formulations disclosed herein may be used in a variety of applications including but not limited to cell culture channels / selection, reorganization, incorporation of mutations into infectious clones, reverse genetics, insertion, deletion, chimeric constructs, , ≪ / RTI > which is associated with a virus that is alive or live and attenuated in any way. In addition, those skilled in the art will appreciate that certain alpha viruses that may benefit from the protective effect of the formulations disclosed herein may be engineered to express other proteins or RNA, including, but not limited to, recombinant alphavirus constructs ≪ / RTI > These viruses include vaccines for infectious diseases, vaccines for treating or reducing the onset of oncological conditions, or proteins or RNAs for treating or preventing conditions, such as gene therapy, antisense therapy, ribozyme therapy or small inhibitory RNA Lt; RTI ID = 0.0 > expression). ≪ / RTI >

In some embodiments, the compositions disclosed herein may contain one or more viruses ( e. G., Enveloped viruses) that have a membrane envelope of tobacco virus, or coronavirus, or any alpha virus of the tovirus family have. In other embodiments, the compositions herein may contain a positive virus, or a positive strand RNA virus, having one or more envelopes of the cornavirus family. In certain embodiments, the composition comprises one or more live, attenuated alphaviruses having one or more insertions, deletions or mutations (e.g., Chikunguniya, VEEV, EEEV, etc.) to induce attenuation of the virus for use in the vaccine composition Or the like).

In certain embodiments, a live alpha virus composition can include one or more live, attenuated alphavirus constructs or viruses as described below: U.S. Application No. PCT / US2009 / 000458, filed January 23, 2009 Filed on July 23, 2010, both applications and their successive applications and divisional applications, are incorporated herein by reference in their entireties for all purposes, and are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to the field of computer- .

Pharmaceutical composition

Some embodiments herein are directed to pharmaceutical compositions for attenuated viral living in aqueous or lyophilized form. Those skilled in the art will appreciate that formulations that reduce freeze-thaw inactivation of the pharmaceutical compositions disclosed herein and that improve thermal viral stability may be in powder form, in freeze-dried or lyophilized form, ≪ / RTI > it is possible to improve the product produced by the process. After reconstitution, such stabilized immunogenic compositions may be administered by a variety of routes including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, pulmonary or oral administration.

In other embodiments, devices for delivering any of the pharmaceutical compositions disclosed herein are contemplated. According to these embodiments, a variety of devices may be used, including but not limited to, syringe and needle injection, branched needle administration, intravenous patch or pump administration, intradermal needle-free jet delivery (intra-dermal) , Or delivery of a vaccine or other immunogenic composition comprising aerosol powder delivery.

In certain embodiments, the compositions disclosed herein typically comprise a physiologically acceptable buffer. In some embodiments, the HEPES buffer has been found to have unexpected stabilizing effects in the alpha virus composition when compared to other buffers. In certain embodiments, buffered media having an over pH of 6.0 to about pH 10 are contemplated; Some live, attenuated or live viruses (eg, alpha viruses) are unstable at low pH.

Embodiments herein provide administration of a composition to a subject in a biologically compatible form suitable for pharmaceutical administration in vivo . &Quot; Biologically compatible form suitable for in vivo administration " means a form of active agent ( e . G., A live, attenuated virus of a particular embodiment) such that any toxic effect is administered at a point governed by the therapeutic benefit of the active agent can do.

Administration of a therapeutically active amount of a pharmaceutical composition disclosed herein may be defined as an effective amount for a period of time necessary to achieve a desired result and at a dosage. For example, the therapeutically effective amount of the compound may vary depending on factors such as the disease state of the individual, age, sex, and weight, and the ability to form the desired response in the individual. Dosage regimens may be adjusted to provide optimal therapeutic response of a particular individual.

In some embodiments, the composition ( e.g., pharmaceutical) can be administered to a subject in any known manner, for example, by subcutaneous, intramuscular, intravenous, intradermal, oral, inhalation, transdermal, Topical, intranasal or rectal administration. In one embodiment, the compound can be administered intranasally, e.g., by inhalation. In certain embodiments, the immunogenic compositions disclosed herein can be administered subcutaneously or intradermally.

The compounds or active agents disclosed herein (e. G., Live, attenuated alpha viruses) may be administered to a subject in a suitable carrier or diluent co-administered with the composition. The term " pharmaceutically acceptable carrier " as used herein is intended to include diluents such as saline and aqueous buffer solutions. The active agent may also be administered parenterally or intraperitoneally.

Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (if water soluble) or dispersions and sterilized powders for the instant preparation of sterile injectable solutions or dispersions may be used. In all cases, the composition may be sterile and may be fluid to the extent that easy needle penetration is real. Can be additionally preserved against the contaminating action of microorganisms such as bacteria and fungi. Pharmaceutically acceptable carriers include, for example, solvents or dispersions containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof, Can be every day. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Upon formulation, the solution may be administered in such an amount that is compatible with the dosage formulation and therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above. In some embodiments, the formulations disclosed herein may be administered before, during, and / or after exposure to the alpha viruses of the invention.

Kit

Other embodiments are directed to kits for use in the compositions and methods disclosed herein. Compositions and live viral formulations may be provided in kits for use in scientific research or for delivery and / or use for administration to a subject. According to these embodiments, the kit may include, but is not limited to, a suitable container, an alpha virus or a live, attenuated alpha virus composition and one or more additional agents such as other anti-viral agents, antifungal or antibacterial agents.

In other embodiments, the kit may further comprise a suitably fractioned alpha virus composition disclosed herein. Also, the composition herein may be partially or totally dehydrated or aqueous. The kits contemplated herein may be stored at room temperature or at a refrigerated temperature, as described herein, depending on the particular formulation.

In other embodiments, the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container from which the composition may be placed and suitably dispensed. Where additional ingredients are provided, the kit may also generally contain one or more additional containers into which such a preparation or ingredient may be placed. The kits herein may also include those containing a composition in a commercial container and any other reagent. Such containers may include injection or blow-molded plastic containers in which the desired vial is retained ( e.g., an immunogenic or vaccine composition).

Example

The following examples are included to demonstrate certain embodiments presented herein. It will be appreciated by those skilled in the art that the techniques disclosed in the following embodiments may be regarded as representing techniques discovered to function well in the practice disclosed herein and thus constitute a preferred manner for its practice . However, those skilled in the art should appreciate that in light of the present disclosure, many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure .

Example 1

Buffer screen

In certain exemplary methods, the use of liquid compositions and lyophilizable compositions and alpha virus immunogenic compositions or vaccines suitable for preclinical and clinical trials are identified. One consideration with liquid compositions according to these exemplary compositions is that some of the alphaviruses are pH sensitive ( e.g., at low pH). Thus, the components of the compositions disclosed herein contain careful consideration of pH. In certain exemplary compositions, the pH of the formulation was about pH 6.0 to about 10.0 with several formulations maintained at about pH 6.5 to 7.5.

In some methods, live, attenuated Chikunguniya virus (CHIK) is used as an example of an alpha-viral composition for pre-clinical and clinical use. Compositions for these methods are provided. In one exemplary experiment, a predetermined amount of CHIK-IRES vaccine (pMVS), which is a live, attenuated Chichungniyya virus with structural genes under inserted IRES control, is used. It is contemplated that any attenuated or live alpha virus may be used in these exemplary compositions to increase the stability of the alpha virus and to reduce degradation. Initially, many different buffers such as DMEM, PBS, HEPES and others were tested.

In another exemplary method, incubation was performed for up to 6 weeks at 40 < 0 > C to test the stability of a particular test, e.g., live, attenuated virus. Samples were obtained to titrate against the presence of infectious virus with TCID ₅₀ in 96 well plates of Vero cells. Percentage of residual virus was calculated when compared to injected (non-incubated) alpha virus composition control. For example, incubation of 10 ⁵ TCID ₅₀ of live alphavirus containing PBS alone, 20% FBS DMEM or DMEM buffered dextrose demonstrated rapid loss in the efficacy of the alpha virus (e.g., CHIK). Certain exemplary compositions comprise HEPES buffer ( 4- (2-hydroxyethyl) phosphate) in the presence of live attenuated alpha viruses such as live, attenuated alpha viruses (e.g., CHIK), e.g., about 1.0 to about 200 mM HEPES . -1-piperazine ethanesulfonic acid ) (data not shown). In one example, a composition containing 15 mM HEPES (HEPES buffered saline - HS) and 150 mM NaCl was found to provide increased stability to attenuated alpha virus vaccine when compared to the control (data not shown).

Example 2

Screening for formulation stabilization without protein allergen

In certain methods, some formulations use hydrolyzed gelatin instead of serum albumin as a protein source (Burke, Hsu et al. 1999) and the previously presented invention relates to the present invention. However, gelatin can cause allergic reactions when administered to a subject, providing stability to live, attenuated viruses, and can sometimes cause vaccine-related adverse events. Certain compositions disclosed herein may be formulated as a combination of ingredients that, when administered to a subject, provide a formulation with a reduced allergic response while providing results comparable to those containing gelatin to reduce the deterioration of live, attenuated alpha virus . ≪ / RTI >

In some instances, formulations that do not have albumin or gelatin that are capable of providing comparable or even increased stability to live alpha viruses or live, attenuated alpha viruses are described. These formulations include, but are not limited to, HEPES buffer, one or more carbohydrate preparations, one or more amino acids, one or more protein preparations, and / or one or more salt formulations.

An exemplary formulation is provided below (all formulations tested below were about pH 7.2 +/- 0.2):

It has been demonstrated that I. 150 mM HEPES, 5% sucrose, 1% gelatin, and 0.9% sodium chloride (positive control) 15.0 mM and 150.0 mM HEPES behaved similarly for alpha virus stabilization.

II. 7% sucrose, 3% human serum albumin, and 1.5% mannitol (protein containing potential allergen)

III. 2.5% sucrose, and 3% histidine

IV. 1.5% sucrose, and 2% glycine

V. 1.5% sucrose, 2% glycine, 0.44% sodium chloride

VI. 2% sucrose, 4% mannitol, 0.5% histidine

VII. 5% sucrose, 0.64% sodium chloride, 0.1% monosodium glutamate, 0.09% sodium phosphate, 0.016% potassium phosphate, and 0.016% potassium chloride

VIII. 2.5% mannitol, and 4.5% peptone

Live, attenuated alpha virus samples formulated in these compositions were stored at controlled temperatures. Samples 18, 15 and 9 per formulation were stored at either 4 ° C, 25 ° C, and 40 ° C, respectively. Samples were observed for efficacy evaluation at the time indicated in Table 1 and illustrative Figures 3-5. Samples incubated at 25 ° C and 4 ° C (Figures 4-5, respectively) were analyzed in parallel with samples incubated at 40 ° C to observe stability trends over a 4 to 26 week period (Figure 3).

Table 2 shows the compilation of exemplary results from experiments using Formulations I-VIII at Week 0. Physiological features such as mean resuspension time, pH, cake appearance score, appearance description, anticipated osmosis, and residual moisture were recorded and recorded for each formulation. Formulations I, II, and VIII were compared to formulations containing protein preparations (e.g., gelatin, albumin, and peptone) and used as controls and not containing protein formulations. For example, Formulations III, VI, and VII did not contain protein and exhibited a cake appearance score of greater than 3 and had a moisture percentage of about 0.47% to about 1.15%.

Table 2 List of Formulations for Study I

Example 3

Liquid and lyophilized formulations

In another exemplary method, the stability of lyophilized attenuated alpha viral formulations ( e.g., CHIK) was assessed. None of the formulations lost significant titer during lyophilization at week 0. Formulations V and VIII, however, lost significant titer during processing, an indicator of liquid stability (see FIG. 1). In addition, Formulation V showed a significant loss of potency in the freeze-thaw cycle of the liquid formulation (Figure 2). The lyophilized Formulations I-VIII were stored at 40 DEG C (FIG. 3), 25 DEG C (FIG. 4), or 4 DEG C (FIG. Samples obtained at the indicated time points were reconstituted and titrated in Vero cells with TCID ₅₀ . (2% sucrose, 4% mannitol, 0.5% histidine) and Formulation VII (5% sucrose, 0.64% sodium chloride, 0.1% monosodium glutamate, , 0.09% sodium phosphate, 0.016% potassium phosphate, and 0.016% potassium chloride) demonstrated stabilization features comparable to those of control formulations I and II for 4 weeks at 40 ° C (FIG. 3). These formulations also demonstrated minimal loss of viral titer over 25 weeks at < RTI ID = 0.0 > 25 C < / RTI > and over 12 weeks (Figures 4 and 5).

Figure 6 is a histogram plot illustrating the loss of titer during accelerated and real-time stability experiments. All formulations were tested for stability given to live attenuated virus at 4 ° C from week 1 to week 26, whereas Formulations IV, V, and VIII lost significant titers after 4 weeks at 40 ° C. Formulations < RTI ID = 0.0 > VI < / RTI > and VII provided stabilizing compositions to live attenuated virus at 40, 25, Formulation VI (2% sucrose, 4% mannitol, 0.5% histidine) was selected as a candidate for further evaluation and expansion.

Example 4

Stabilized formulations without human and mammalian protein preparations

In other exemplary methods, long-term stability experiments at 40 ° C, 25 ° C, and 4 ° C have shown that carbohydrates (eg, sucrose and mannitol) and amino acids (eg, histidine) For example, live, attenuated CHIK was used based on the observation that formulations with more than one carbohydrate and more than one amino acid had a positive effect on CHIK stability (see, for example, See above). Using the formulation VI presented above as the base formulation, formulations with varying concentrations of sucrose, mannitol, and histidine were formulated with 15 mM HEPES buffer at pH 7.2 +/- 0.2.

Exemplary formulations:

1. 15.0 mM HEPES, 25.0 mg / ml mannitol, 12.5 mg / ml histidine, and 35.0 mg / ml sucrose

2. 15.0 mM HEPES, 25.0 mg / ml mannitol, 12.5 mg / ml histidine, and 20.0 mg / ml sucrose

3. 15.0 mM HEPES, 40.0 mg / ml mannitol, 5.0 mg / ml histidine, and 50.0 mg / ml sucrose

4. 15.0 mM HEPES, 25.0 mg / ml mannitol, 20.0 mg / ml histidine, and 35.0 mg / ml sucrose

5. 15.0 mM HEPES, 40.0 mg / ml mannitol, 5.0 mg / ml histidine, and 20.0 mg / ml sucrose (equivalent to Formulation VI discussed above)

6. 15.0 mM HEPES, 25.0 mg / ml mannitol, 12.5 mg / ml histidine, and 35.0 mg / ml sucrose

7. 15.0 mM HEPES, 40.0 mg / ml mannitol, 20.0 mg / ml histidine, and 20.0 mg / ml sucrose

8. 15.0 mM HEPES, 25.0 mg / ml mannitol, 5.0 mg / ml histidine, and 35.0 mg / ml sucrose

9. 15.0 mM HEPES, 25.0 mg / ml mannitol, 12.5 mg / ml histidine, and 50.0 mg / ml sucrose

10. 15.0 mM HEPES, 40.0 mg / ml mannitol, 20.0 mg / ml histidine, and 50.0 mg / ml sucrose

11. 15.0 mM HEPES, 10.0 mg / ml mannitol, 20.0 mg / ml histidine, and 20.0 mg / ml sucrose

12. 15.0 mM HEPES, 10.0 mg / ml mannitol, 12.5 mg / ml histidine, and 35.0 mg / ml sucrose

13. 15.0 mM HEPES, 10.0 mg / ml mannitol, 20.0 mg / ml histidine, and 40.0 mg / ml sucrose

14. 15.0 mM HEPES, 10.0 mg / ml mannitol, 5.0 mg / ml histidine, and 20.0 mg / ml sucrose

15. 15.0 mM HEPES, 40.0 mg / ml mannitol, 12.5 mg / ml histidine, and 35.0 mg / ml sucrose

16. 15.0 mM HEPES, 10.0 mg / ml mannitol, 5.0 mg / ml histidine, and 40.0 mg / ml sucrose

15.0 mM HEPES buffered saline (positive control) containing sucrose and gelatin (HSG) 15.0 mM HEPES, 5% sucrose, 1% gelatin, and 0.9% sodium chloride. It was demonstrated that 15.0 mM and 150.0 mM HEPES behaved similarly for alpha virus stabilization.

Live, attenuated alpha virus samples formulated in these compositions were stored. 15 samples per formulation were stored at 4 ° C, 25 ° C, or 40 ° C. Sample formulations were studied for efficacy evaluation at the time indicated in Table 3 and exemplary results were described in Figures 10-12. The samples incubated at 4 째 C and 25 째 C (Figs. 11-12) were analyzed in parallel with the samples incubated at 40 째 C to demonstrate the trend of remaining titer for each 12 and 6 week periods, respectively (Fig. 10).

Example 5

Liquid and lyophilized formulations

In another exemplary method, the stability of lyophilized live, attenuated alpha viruses (e. G., CHIK) was assessed. At 0 weeks, all compositions 1-16 were analyzed for physiological features and showed a cake appearance score of greater than 3 and had a percent moisture content of about 0.46% to about 1.46% (data not shown).

Some of these formulations showed higher residual potency when lyophilized at 0 weeks (Figure 8). Some of these formulations exhibited higher residual potency in the freeze-thaw cycle (Figure 9). In another experiment, lyophilized vaccine Formulations 1-16 were stored at 40 캜 (Fig. 10), 25 캜 (Fig. 11), or 4 캜 (Fig. 12) and evaluated for overall potency loss at various temperatures. Samples obtained at the indicated time points were reconstituted and titrated in Vero cells using the indicator to calculate TCID ₅₀ . Exemplary living alpha viruses (e. G. Live, attenuated CHIK) Formulations 1, 3, 9, and 10 of this test group compared to the positive control group 17 were stable at < , While other Formulations 2, 5, 7, 11, 12, 13 and 14 were found to be at room temperature when compared to 1, 3, 9, 10 and Control Formulation 17, at least 6 weeks when stored at about 40 캜 (FIG. 10) , Respectively. Formulations 1, 3, 9, and 15 compared to positive control formulation 17 were stable for at least 12 weeks when stored at about 25 캜, while Formulations 5, 11, and 13 showed significant potency loss (Fig. 11). Despite this comparable loss at 25 캜, all of the formulations (1-16) tested demonstrated minimal loss of viral titer over 12 weeks when stored at a refrigeration temperature of about 4 캜 (Fig. 12).

Figure 13 is a histogram plot illustrating the loss of activity during the stability studies described above. It was observed that all formulations maintained a stable titer of live alpha virus at a refrigeration temperature of about 4 < 0 > C for at least 12 weeks. At room temperature, approximately 40 占 폚, Formulations 2, 5, 7, 11, 12, 13, and 14 showed significant loss by about 6 weeks. Formulations 1, 3, 9, 10, and 15 demonstrated improved stability compared to Formulation 5 (equivalent to Formulation 6 in Example 1 above) for periods of longer than 6 weeks at room temperature, about 40 캜.

Figure 14 shows an output illustrating data from experiments using various formulations to predict desirable properties in an exemplary live alpha virus formulation. These data provide support that formulations with high concentrations of certain carbohydrate preparations and low concentrations of specific amino acids showed improved stability over several weeks at room temperature of about < RTI ID = 0.0 > 40 C. < / RTI > Thus, these specified formulations may be used to increase the stability of live alpha viruses during transportation, delivery and final administration to a subject. In addition, these exemplary formulations are free of human or animal protein formulations that can reduce negative immunity and other effects in the recipient.

Example 6

Formulation stabilization without human or animal-derived protein preparation

In other exemplary methods, long-term stability experiments at 40 ° C, 25 ° C, and 4 ° C have shown that additional various concentrations of carbohydrates (eg, sucrose and mannitol) and amino acids (eg, histidine) For example live, attenuated CHIK viruses were used based on the observation that formulations with more than one carbohydrate and more than one amino acid had a positive effect on CHIK stability remind). Using the formulation VI presented above as the base formulation, formulations with various concentrations of sucrose, mannitol, and histidine were formulated with 15.0 mM HEPES buffer at pH 7.2 +/- 0.2. In addition, the swelling of these target preparations, including compositions of mannitol, histidine, and sucrose, was based on observations in the above 17 sample studies. Mannitol, sucrose, histidine, and HEPES were selected in combination in a 17 formulation study and based on its performance against its non-animal origin. The target concentrations selected for mannitol and sucrose in this group of 10 formulations were based on statistical predictions using data generated in previous experiments.

Exemplary formulations:

1. 15.0 mM HEPES, 25.0 mg / ml mannitol, 5.0 mg / ml histidine, and 50.0 mg / ml sucrose

2. 15.0 mM HEPES, 35.0 mg / ml mannitol, 5.0 mg / ml histidine, and 30.0 mg / ml sucrose

3. 15.0 mM HEPES, 15.0 mg / ml mannitol, 5.0 mg / ml histidine, and 30.0 mg / ml sucrose

4. 15.0 mM HEPES, 25 mg / ml mannitol, 5.0 mg / ml histidine, and 50.0 mg / ml sucrose

5. 15.0 mM HEPES, 35.0 mg / ml mannitol, 5.0 mg / ml histidine, and 70.0 mg / ml sucrose

6. 15.0 mM HEPES, 25.0 mg / ml mannitol, 5.0 mg / ml histidine, and 21.72 mg / ml sucrose

7. 15.0 mM HEPES, 10.86 mg / ml mannitol, 5.0 mg / ml histidine, and 50.0 mg / ml sucrose

8. 15.0 mM HEPES, 25.0 mg / ml mannitol, 5.0 mg / ml histidine, and 78.28 mg / ml sucrose

9. 15.0 mM HEPES, 15.0 mg / ml mannitol, 5.0 mg / ml histidine, and 70.0 mg / ml sucrose

10. 15.0 mM HEPES, 39.14 mg / ml mannitol, 5.0 mg / ml histidine, and 50.0 mg / ml sucrose

Example 7

Liquid and lyophilized formulations

Live, attenuated alpha virus samples formulated in these compositions were stored. In this study, live, attenuated Chikunguniya virus stability in various formulations based on 17 sample studies was analyzed. In this study, Sample 17 was selected as a positive control. 15 samples per formulation were stored at 4 占 폚, 25 占 폚, or 40 占 폚. Sample formulations were studied for efficacy evaluation at the time indicated in Table 4 and exemplary results were described in Figures 16-20. Samples incubated at 4 占 and 25 占 (FIGS. 19 and 18, respectively) were analyzed in parallel with samples incubated at 40 占 폚 in order to demonstrate the trend of remaining titer over a 6-12 week period (FIG. 17).

Example 8

In another exemplary method, the stability of lyophilized live, attenuated alpha viruses (e. G., CHIK) was assessed. At Week 0, all Compositions 1-10 were analyzed for physiological features and showed a cake appearance score of greater than 3 and had a <0.5% moisture remaining percentage (data not shown). A center-point formulation (Rx 1 and 4 in this study) containing 25 mg / ml mannitol and 50 mg / ml male cross was able to achieve higher stability during formulation at both 25 ° C and 40 ° C, both temperatures critical for stability during transport And met the positive physical criteria for cake aesthetics (data not shown). In one example, these samples were duplicated to generate more robust statistical data set for the excipient concentration center point of the experimental design.

Some of these formulations exhibited higher residual titer when lyophilized at week 0 (Figure 16). For example, 1, 4, 5, 8 and 9 demonstrated higher titer in liquid form. On the other hand, Formulations 5 and 8 demonstrated higher titer in lyophilized condition (Figure 16). Some of these formulations exhibited higher residual titer in the freeze-thaw cycle (data not shown). In other experiments, lyophilized vaccine Formulations 1-10 were stored at 40 占 (FIG. 17), 25 占 (18), or 4 占 (19) and evaluated for overall potency loss at various temperatures during specified times . Samples obtained at the indicated time points were reconstituted and titrated in Vero cells using the indicator to calculate TCID ₅₀ . Examples 1, 3, 4, 5, 8, 9, and 10 of exemplary live alpha viruses (e. G., Live, attenuated CHIK virus) of this test group were tested for> 60% While other Formulations 2, 6, and 7 showed slightly reduced recovery rates for at least 6 weeks when stored at room temperature of about 40 ° C as compared to Formulations 1, 3, 4, 5, 8, 9, (Fig. 17). All tested formulations had a recovery of at least 50% for at least 6 weeks after exposure to room temperature of about &lt; RTI ID = 0.0 &gt; 40 C &lt; / RTI & This is a significant improvement over other formulations tested in the art and herein.

Formulations 1, 2, 4, 5, 8, and 10 demonstrated at least 80% recovery for at least 12 weeks when stored at about 25 ° C while Formulations 3, 6, 7, 9, Week and had some potency loss when compared to other tested formulations (Figure 18). Despite this comparable loss at 25 占 폚, all formulations (1-10) tested demonstrated at least 70% recovery for at least 12 weeks if stored at about 25 占 (FIG. 18). All formulations tested (1-10) demonstrated only slight loss and had an over 90% recovery when stored at refrigeration temperature of about 4 ° C for at least 12 weeks (Figure 19).

Figure 20 is a histogram plot illustrating the loss of activity during the stability studies described above. It was observed that all formulations maintained a stable titer of live alpha virus at a refrigeration temperature of about 4 &lt; 0 &gt; C for at least 12 weeks. At room temperature, approximately 40 占 폚, Formulations 2, 3, 6 and 7 showed significant loss by about 6 weeks. Formulations 1, 8, and 9 demonstrated improved stability compared to Formulation 2 for periods of longer than 6 weeks at room temperature, about 40 &lt; 0 &gt; C.

Materials and methods

A separate aliquot of the predetermined volume of CHIK-IRES vaccine (pre-MVS, master virus seed) contains HEPES buffered saline containing HEPES buffered saline (HB), sucrose (HBS), sucrose and gelatin 0.0 &gt; HEPES &lt; / RTI &gt; buffered saline. The formulated hydrated or liquid vaccine was incubated at specific temperatures, such as room temperature 25 占 폚 and 40 占 폚, and refrigeration temperature 4 占 폚. Aliquots were removed from these formulations at predetermined intervals and titrated for the presence of infectious alpha viruses, for example, with TCID ₅₀ in 96 well plates with Vero cells.

Cell line and tissue culture

A study-grade Vero cell bank derived from the applicant's cGMP Working Cell Bank was prepared to perform these experiments. Vero cells were obtained and stored in liquid nitrogen: Vero (WHO) Working Cell Bank passage: 142 (lot # INV-VERO-WCB-001; 5x10 ⁶ ). The vials were pre-warmed cDMEM (Dulbecco's modified minimal essential medium), which was rapidly thawed in aquarium and contained penicillin-streptomycin, 40 mM L-glutamine and 10% FBS in T-75 cm ² flasks, Inoculated and incubated at 37 ° C, 5% CO ₂ . The cells grew in density and were subcultured using PBS, trypsin (Hyclone, e.g. cat # SH30042.01) and cDMEM-10. The flasks were expanded to 2 T-185 cm ² flasks and grown until the cells reached 100% density. Cells were harvested by trypsinization and centrifuged at 800 xg for 10 minutes and resuspended in DMEM containing 20% FBS and 10% DMSO at a concentration of 1x10 ⁷ cells / mL.

Vero was grown and maintained in Dulbecco's Modified Minimum Essential Medium (DMEM) (DMEM-10% -FBS) containing penicillin-streptomycin and 10% FBS (Hyclon). Trypsin was used to maintain the cells. Virus adsorption 2 days ago, 96-well plates at 100 uL per well of DMEM-FBS-10% 1.4 x 10 5 cells were plated in / mL. The incubator was monitored daily to maintain the specified temperature. Virus dilution, adsorption and TCID50 assays were performed in 2% cDMEM-FBS.

CHIK-IRES: live, attenuated alpha virus

Molecular generation of the live, attenuated CHIK virus used in the various methods described is designated CHIK-002 (previously described). CHIK-002 was produced by IRES insertion and propagated in Vero cells. The pre-master virus seed stock was used in these experiments at a concentration of 10 ⁵ TCID ₅₀ / mL. Briefly, CHIK pre-MVS was generated after infection of a single layer of Vero cells. The CHIK-virus is secreted into the supernatant and the virus is harvested from the medium after purification / removal of the dead Vero cells. CHIK-pre-MVS was stabilized in DMEM containing 10% FBS and stored at -80 ° C.

Black method

The TCID ₅₀ assay method was used to quantify the amount (potency or stability) of the infectious virus present in a virus-containing preparation. TCID ₅₀ is defined as the level of dilution of the virus showing that half of a series of replicates of infected wells in a 96-well plate is indicative of viral infectivity, as evidenced by, for example, CPE (cytopathic effect) . Vero cells were grown and maintained in Dulbecco's Modified Minimum Essential Medium (DMEM) (DMEM 10% -FBS) containing penicillin-streptomycin, L-glutamine and 10% FBS (Hyclon). The aliquots of the formulated samples were rapidly thawed and mixed in a water bath. Initial dilutions of pre-MVS were performed at working concentrations, and a 10-fold dilution series of these samples was made, for example, in cDMEM-2% FBS in 96-well plates. The diluted virus was maintained at 4 ° C prior to inoculation of the Vero cell monolayer. At the time of assay, the growth medium was aspirated from the 96-well plate, and 100 μL of each of the virus dilutions was added to the wells. Plates were incubated at 37 ℃ and 5% CO ₂ for 3-5 days. The station was calculated using the Spearman-Carver method.

Vaccine formulation

Stability experiments were prepared with a vaccine containing the study-grade vaccine formulation, and internally derived CHIK-IRES pMVS. For screening of excipients and stability studies using the various compositions provided herein, alphavirus formulations were prepared in a final volume of 500 μL containing 10 ⁵ TCID ₅₀ / mL virus per sample. Samples were prepared in bulk in the specified buffers / formulations and input samples were obtained before the study was initiated as a measure of initial potency. Samples were aliquoted into MCT and stored for the indicated time and temperature. Each of the four formulations was prepared in 500 μL final with 10 ⁵ TCID ⁵⁰ / mL virus per sample. Sixty samples per formulation were made in bulk and the input samples were obtained before they were aliquoted into 1.5 mL MCT containing 500 uL.

Store formulated immunogenic constructs

Immunogenic viral formulations were stored at 4 ° C (Micro Climate Chamber; Model # MCB-12-33-33-H / AC) and at -80 ° C (REVCO Elite Plus; Model # ULT2186-6-D43). Both systems were monitored with the Dickson Wizard2 - 900MHZ Logger (Model # WT-220 for 4 ° C and WT-240 for -80 ° C).

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods are described herein with respect to preferred embodiments, it is contemplated that changes may be made to the compositions and methods in the steps, or in the sequence of steps, of the methods described herein without departing from the concept, spirit and scope herein It is obvious to a skilled person. More specifically, certain agents, both chemically and physiologically related, may be substituted with the agents described herein while the same or similar results are achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims.

Claims (62)

A live attenuated alphavirus composition comprising:
One or more live alpha viruses;
One or more amino acids including histidine;
At least two carbohydrate preparations;
Wherein the composition does not comprise serum or gelatin, and wherein the composition stabilizes the live alpha virus. The method according to claim 1,
Further comprising a HEPES buffer, wherein the HEPES buffer concentration is from 1.0 mM to 200.0 mM. The method according to claim 1 or 2,
Wherein said one or more live alphaviruses are selected from the group consisting of Chikungunya virus (CHIK), sour virus, Los River virus, eastern encephalitis and western encephalitis, other sickle wood viruses or other tooviruses, Alpha virus composition. The method according to claim 1 or 2,
Wherein said at least one live alphavirus is chikunguniya (CHIK) virus. The method according to any one of claims 1 to 4,
Wherein said live alpha virus comprises live attenuated alpha virus. The method according to any one of claims 1 to 5,
Wherein said composition is in aqueous form. The method according to any one of claims 1 to 5,
Wherein said composition is partially or totally dehydrated. The method according to any one of claims 1 to 7,
Wherein the two or more carbohydrate formulations are selected from the group consisting of one or more of trehalose, galactose, furutose, sucrose, chitosan, sorbitol, mannitol, and combinations thereof. The method according to any one of claims 1 to 8,
Wherein said at least two carbohydrate preparations comprise sucrose and mannitol. The method according to any one of claims 2 to 9,
Wherein the concentration of the HEPES buffer concentration is 10.0 mM to 150.0 mM. The method according to any one of claims 2 to 10,
The HEPES buffer concentration is 10.0 mM to 30.0 mM; Wherein the two or more carbohydrate preparation concentrations are 10.0 to 100.0 mg / ml; And the amino acid concentration is 1.0 mg / ml to 20.0 mg / ml. The method according to any one of claims 2 to 11,
The HEPES buffer concentration is 15.0 mM; Wherein the two or more carbohydrate preparation concentrations are 20.0 mg / ml to 70.0 mg / ml, respectively; And the amino acid concentration is from 1.0 mg / ml to 10.0 mg / ml. The method according to any one of claims 2 to 12,
Wherein said alpha virus composition comprises HEPES buffer, histidine, sucrose, and mannitol. 14. The method of claim 13,
The HEPES buffer concentration is 15.0 mM and the histidine concentration is 1.0 mg / ml to 20.0 mg / ml; The sucrose concentration is 20.0 mg / ml to 90.0 mg / ml; And the mannitol concentration is 10.0 mg / ml to 50.0 mg / ml. The method according to claim 13 or 14,
The HEPES buffer concentration is 15.0 mM and the histidine concentration is 5.0-20.0 mg / ml; The sucrose concentration is 40.0 to 90.0 mg / ml; And mannitol is 15.0 to 40.0 mg / ml. 16. The method of claim 15,
The histidine concentration is 5.0 to 15.0 mg / ml; The sucrose concentration is 40.0 to 80.0 mg / ml; And the mannitol concentration is 15.0 to 40.0 mg / ml. The method according to any one of claims 1 to 16,
&Lt; / RTI &gt; further comprising a salt. 22. A live attenuated alphavirus composition, comprising:
One or more live alpha viruses;
One or more salt formulations; And
One or more carbohydrate formulations comprising sucrose;
Wherein the composition does not comprise serum or gelatin or other mammalian protein product, and wherein the composition stabilizes the live alphavirus. 19. The method of claim 18,
Further comprising a HEPES buffer, wherein the HEPES buffer concentration is from 1.0 mM to 200.0 mM. The method according to claim 18 or 19,
Wherein said one or more live alphaviruses are selected from the group consisting of Chikungunya virus (CHIK), sour virus, Los River virus, eastern encephalitis and western encephalitis, other sickle wood viruses or other tooviruses, Alpha virus composition. The method according to claim 17 or 18,
Wherein said at least one live alpha virus comprises a live attenuated alpha virus. The method according to any one of claims 18 to 21,
Wherein the at least one live alpha virus is a live chickens virus (CHIKV). The method as claimed in any one of claims 18 to 22,
Wherein said composition is in aqueous form. 23. The method according to any one of claims 18-23,
Wherein said composition is partially or totally dehydrated. The method of any one of claims 18 to 24,
Wherein the composition further comprises at least one additional carbohydrate preparation selected from the group consisting of mannitol, trehalose, galactose, furutose, chitosan, sorbitol, mannitol, and combinations thereof. The method of any one of claims 18 to 25,
Wherein said at least one salt preparation is selected from the group consisting of sodium chloride, monosodium glutamate (MSG), sodium phosphate, potassium phosphate, potassium chloride, and combinations thereof. 26. The apparatus according to any one of claims 19 to 26,
Wherein the HEPES concentration is from 1.0 mM to 40.0 mM. 28. The method according to any one of claims 19 to 27,
The HEPES buffer concentration is 1.0 to 30.0 mM; Wherein the at least one carbohydrate preparation concentration is 1.0% to 10.0%; And each said at least one salt concentration is between 0.001% and 1.0% (w / v). The method of any one of claims 18 to 26,
Wherein said composition comprises HEPES, sucrose, sodium chloride, monosodium glutamate, sodium phosphate, potassium phosphate, and potassium chloride. 29. The method of claim 29,
The HEPES buffer concentration is 15 mM and the sucrose concentration is 1.0% to 15.0% (w / v); The sodium chloride concentration is between 0.1% and 1.5% (w / v); The concentration of monosodium glutamate is 0.05% to 0.5% (w / v); The sodium phosphate concentration is 0.01 to 0.15% (w / v); The potassium phosphate concentration is between 0.01% and 0.1% (w / v); And the potassium chloride concentration is from 0.01% to 0.1% (w / v). Two or more carbohydrate formulations; And combining at least one living alpha virus with a composition comprising at least one amino acid comprising histidine, wherein the composition reduces the inactivation of the live alpha virus. 32. The method of claim 31,
Further comprising a HEPES buffer, wherein the HEPES buffer concentration is from 1.0 mM to 200.0 mM. 32. The method of claim 31 or 32,
Wherein said live alphavirus comprises Chicken &lt; RTI ID = 0.0 &gt; viral &lt; / RTI &gt; virus (CHIK), Sourgan virus, Ross River virus, other Semliki wood heat virus complex, eastern encephalitis and western encephalitis and combinations thereof. 34. The method according to any one of claims 31 to 33,
Further comprising dehydrating said combination partially or wholly. 34. The method according to any one of claims 31 to 33,
Further comprising re-hydrating the composition partially or totally prior to administration. 34. The method according to any one of claims 31 to 35,
Wherein the composition increases the shelf life of the aqueous virus composition. 35. The method of any one of claims 31-36,
The HEPES buffer concentration is 1.0 to 30.0 mM; Wherein the two or more carbohydrate formulation concentrations are each 1.0 to about 100.0 mg / ml; And the amino acid concentration is 1.0 to 40.0 mg / ml. 37. The method of any one of claims 32-37,
Wherein said composition comprises HEPES, histidine, sucrose, and mannitol. 42. The method of claim 38,
The HEPES buffer concentration is 15 mM, and the histidine concentration is 5.0 mg / ml to 20.0 mg / ml; The sucrose concentration is 20.0 mg / ml to 90.0 mg / ml; And the mannitol concentration is 10.0 mg / ml to 40.0 mg / ml. 42. The method of any one of claims 31-39,
&Lt; / RTI &gt; further comprising a salt preparation. 40. The method according to any one of claims 31-40,
Wherein said live alpha virus composition is further formulated for use as a medicament for administration to a subject in order to prevent or prevent alpha virus infection. 0.1 mM to 200 mM HEPES buffer; A method for reducing the inactivation of live alphavirus comprising combining a composition comprising at least one salt formulation and at least one carbohydrate formulation comprising at least one live alpha virus, Thereby reducing inactivation. 43. The method of claim 42,
Wherein said live alphavirus comprises Chicken &lt; RTI ID = 0.0 &gt; viral &lt; / RTI &gt; virus (CHIK), Sourgan virus, Ross River virus, other Semliki wood heat virus complex, eastern encephalitis and western encephalitis and combinations thereof. 43. The method of claim 42 or claim 43,
Further comprising dehydrating said combination partially or wholly. 43. The method of claim 42 or claim 43,
Further comprising re-hydrating the composition partially or totally prior to administration. 45. The method according to any one of claims 42 to 45,
Wherein the composition increases the shelf life of the aqueous virus composition. 47. The method of any one of claims 42-46,
Wherein the two or more carbohydrate preparations further comprise at least one of trehalose, galactose, furutose, chitosan, sorbitol, mannitol, and combinations thereof. 47. The method according to any one of claims 42-47,
Wherein said at least one salt preparation comprises one or more of sodium chloride, monosodium glutamate, sodium phosphate, potassium phosphate, potassium chloride, and combinations thereof. 47. The method of any one of claims 42-48,
Wherein the HEPES buffer concentration is from 1.0 mM to 40.0 mM. 47. The method of any one of claims 42-49,
The HEPES buffer concentration is 1.0 to 30.0 mM; Wherein the two or more carbohydrate preparation concentrations are each 1% to 10%; And the salt agent concentration is 0.001% to 0.2% (w / v). 47. The method of any one of claims 42-50,
Wherein the composition comprises HEPES, sucrose, sodium chloride, monosodium glutamate, sodium phosphate, potassium phosphate, and potassium chloride. A kit for reducing the inactivation of live alphavirus, comprising:
At least one container;
Two or more carbohydrate preparations comprising 0.1 mM to 40.0 mM HEPES buffer, sucrose, and one or more amino acids including histidine; And
One or more living alpha viruses. 53. The method of claim 52,
Wherein the composition comprises HEPES, histidine, sucrose, and mannitol. 53. The method of claim 52 or 53,
Wherein the composition comprises a pharmaceutical composition comprising a pharmaceutically acceptable excipient. A kit for reducing the inactivation of live alphavirus, comprising:
At least one container;
0.1 to 40.0 mM HEPES buffer, at least one carbohydrate preparation comprising sucrose, at least one salt formulation comprising at least monosodium glutamate, and
A living alpha virus. 55. The method of claim 55,
Wherein the composition comprises HEPES, sucrose, sodium chloride, monosodium glutamate, sodium phosphate, potassium phosphate, and potassium chloride. The method of any one of claims 52 to 56,
Wherein said live alpha virus comprises live attenuated alpha virus. The method of any one of claims 53 to 57,
Wherein said live alphavirus comprises a chikunguniya (CHIK) virus, a sour virus, a Ross River virus, a Semliki forest heat virus complex, eastern encephalitis and western encephalitis, and combinations thereof. 32. The method according to any one of claims 1 to 30,
Wherein said alpha virus composition is a live attenuated alpha virus composition, and further comprising a pharmaceutically acceptable excipient. 55. The method of claim 59,
A pharmaceutical composition for use in a method of protecting an object from infection resulting from exposure to an alpha virus. Use of a pharmaceutical composition according to claim 59 for the manufacture of a vaccine for protecting a subject against infection resulting from exposure to an alpha virus. 61. A pharmaceutical composition for use as defined in claim 60, wherein said subject is a human, livestock or other raised animal, and a use according to claim 61.

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